We are developing an in-vivo, somatic mutation assay, applicable to both humans and experimental animals, that detects multiple, base-substitution mutations in red blood cell proteins. Current efforts to establish proof-of-principle involve a version of this assay applied to rabbits. The present proposal seeks to extend this approach to humans and to compare somatic mutation levels measured in humans therapeutically exposed to mutagens with those measured in rabbits experimentally exposed to the same agents. The assay method involves measuring the fraction of circulating erythrocytes that contain any one of a specific set of amino-acid-substituted variants of the proteins hemoglobin and spectrin. Such cells are labeled with fluorescently conjugated antibodies specific for the variant proteins and enumerated using automated cytofluorometry. The critical element in this approach is a method for producing polyvalent antisera that can recognize ten or more different, mutationally inducible, amino-acid-substituted variants of either hemoglobin or spectrin. We propose that a protein from one mammal is an immunologic model for a collection of point mutations in the same protein of other mammalian species; e.g., rabbits immunized with human hemoglobin produce polyvalent antisera, each component of which should monospecifically recognize one human-like mutational variant of rabbit hemoglobin. Sera for detecting point mutations in hemoglobin and spectrin in human erythrocytes can be raised by immunizing primates with purified hemoglobin and spectrin from rabbits. These somatic mutation assay methods should enable us to monitor the direct mutational effects of exposure to genetically toxic agents in individual humans and to compare these effects with the results of identical measurements made in experimentally exposed rabbits. Since there is strong evidence that carcinogenisis is closely related to the accumulation of mutations, this assay allows potentially carcinogenic exposures to be compared between humans and rabbits.